Administration of dispersions by infusion

ABSTRACT

Method and apparatus for continuous infusion to a subject of dispersions in which the dispersed phase is susceptible to flotation or sedimentation. The dispersion is controllably delivered from an upper or lower extremity of an essentially vertically positioned. delivery vessel, e.g. syringe  2,  and is then admixed with flushing medium, e.g. from infusion minibag  8,  prior to administration to the subject. Vertical positioning of the delivery device maximises the distance through which flotation or sedimentation may occur, thereby substantially reducing the effects of separation over a given period of time compared to use of a corresponding horizontal delivery vessel such as a syringe placed in a conventional syringe driver.

[0001] This invention relates to the administration of dynamic (i.e. gravity segregating) particulate dispersion systems, e.g. gas-containing diagnostic contrast agents, more particularly to apparatus and a method for the controlled and substantially steady state administration of such dispersions by infusion.

[0002] In the field of ultrasonography it is well known that contrast agents comprising dispersions of gas microbubbles are particularly efficient backscatterers of ultrasound by virtue of the low density and ease of compressibility of the microbubbles. Such microbubble dispersions, if appropriately stabilised, may permit highly effective ultrasound visualisation of, for example, the vascular system and tissue microvasculature, often at advantageously low doses of the contrast agent.

[0003] Gas-containing contrast media are also known to be effective in magnetic resonance (MR) imaging, e.g. as susceptibility contrast agents which will act to reduce MR signal intensity. Oxygen-containing contrast media also represent potentially useful paramagnetic MR contrast agents.

[0004] In the field of X-ray imaging gases such as carbon dioxide may be used as intravascular contrast agents. Moreover, the use of radioactive gases, e.g. radioactive isotopes of inert gases such as xenon, has been proposed in scintigraphy, for example for blood pool imaging.

[0005] Gas-containing ultrasound contrast agents are usually administered intravenously as a single or multiple bolus dosage, leading to a rapid and pronounced but relatively short lived rise in backscatter intensity in respect of blood-perfused tissue and organs as the bolus mixes with surrounding blood and is carried through the circulation system. A plot of backscatter intensity against time therefore shows a relatively narrow and high signal intensity peak; backscatter measurements are normally made during the existence of this peak, although this may give rise to problems in, for example, the imaging of deeper tissue and organs where high backscatter from overlying tissue may cause excessive shadowing during the peak period.

[0006] As discussed in WO-A-9748337, diagnostic artefacts such as shadowing may be reduced by controlling the rate of administration of the contrast agent and/or by administering a flush such as normal saline after administration of the contrast agent. Contrast agent administration rates of 1-8×10⁶ vesicles/kg-sec or 1×10⁻⁷ to 3×10⁻³ cc gas/kg-sec and flush rates of 0.01-2.4 ml/sec are suggested; the contrast agent is typically administered over a period of 5-20 seconds, and any subsequent flush is typically administered over a period in the range 10 seconds to 10 minutes.

[0007] Continuous infusion of ultrasound contrast agents, for example over a period in the range from one minute to one hour, is of potential interest in that it may permit administration of the contrast agent at a rate which minimises diagnostic artefacts such as shadowing and may lengthen the useful time window for imaging beyond the relatively short duration of the backscatter signal peak resulting from passage of a contrast agent bolus.

[0008] Thus, for example, Albrecht et al. in Radiology 207, pp. 339-347 (1998) note that the use of continuous contrast agent infusion to provide prolonged enhancement of Doppler signals is advantageous in that it may permit completion of lengthy imaging procedures such as studies of the renal arteries or peripheral leg veins and may optimise dose effectiveness of the contrast agents, as well as reducing saturation artefacts.

[0009] Administration of contrast agents by infusion may also be useful in procedures based on imaging of contrast agent in the recirculating phase following admixture with the blood pool, as described in WO-A-9908714.

[0010] A problem with the continuous infusion of gas-containing diagnostic contrast agents arises from the tendency of gas-containing components such as microbubbles to float, since this will lead to inhomogeneities forming within vessels such as power-driven syringes which may be used to administer the contrast agent. This may, for example, lead to an increase in microbubble concentration in the upper part of such a vessel and/or to changes in size distribution occurring at various points within the vessel as larger microbubbles float more rapidly than smaller microbubbles.

[0011] A possible solution to this problem is proposed in WO-A-9927981, which discloses powered injector systems comprising a syringe which is subjected to rotational or rocking motion in order to maintain homogeneity within the contents thereof. In specific embodiments the barrel of the syringe is positioned horizontally in contact with wheels or moveable brackets which are capable of alternately rotating the syringe in opposite directions about its longitudinal (i.e. horizontal) axis.

[0012] It will be appreciated that the incorporation of such rotational or other agitational means into syringe driver apparatus necessarily complicates the design and significantly increases the cost of such apparatus, so that there is an ongoing need for apparatus which permits the continuous infusion of gas-containing ultrasound contrast agents or other gravity segregating dispersions while maintaining substantial homogeneity of the contrast agent or other dispersion.

[0013] The present invention is based on the finding that controlled delivery of a substantially homogeneous gravity segregating dispersion may be achieved by an infusion procedure in which the dispersion is delivered from an upper or lower extremity of an essentially vertically positioned delivery vessel, e.g. a syringe, tubing or other cylindrically shaped reservoir, and is admixed with a flushing medium prior to administration to a subject.

[0014] By using an essentially vertically positioned cylindrical delivery vessel such as a syringe, as opposed to the horizontal orientation normally employed in delivery devices such as syringe drivers, the height of the dispersion sample in the vessel is greatly increased, thereby extending the distance through which gravity segregation may occur. Since relatively low density dispersed moieties such as microbubbles or other gas-containing components of a given size will rise through carrier liquid at a constant rate, this significantly reduces the effects of flotation separation and thereby improves dose control over a given period of time. Similar considerations apply to dispersions of relatively high density microparticles, emulsion droplets etc. which tend to sediment over time.

[0015] Co-administration of the dispersion with an admixed flushing medium further enhances product homogeneity, e.g. by reducing the residence time of the dispersion in connecting tubing etc., thereby reducing its susceptibility to gravity segregation. Admixture with flushing medium also permits particularly efficient control of administration of the dispersion since the flow rates of both the dispersion and the flushing medium may be independently controlled.

[0016] Admixture of the dispersion with flushing medium almost immediately prior to administration to a subject is particularly advantageous in the administration of dispersions such as gas microbubble-containing contrast agents, which often show instability if stored in diluted form, e.g. if diluted prior to transfer into a syringe or other delivery vessel.

[0017] Moreover, where administration is by intravascular (e.g. intravenous) injection, coadministration of admixed flushing medium at a single injection site assists in maintenance of an open injection route independent of dispersion flow and local blood flow variations.

[0018] Thus according to one aspect of the present invention there is provided a method of administering a gravity segregating dispersion, e.g. a gas-containing contrast agent, to a subject by continuous infusion, wherein said dispersion is controllably delivered from an upper or lower extremity of an essentially vertically positioned delivery vessel, e.g. a syringe, and thereafter is admixed with a flushing medium prior to administration to the subject.

[0019] According to a further aspect the invention provides apparatus useful in the administration of a gravity segregating dispersion, e.g. a gas-containing contrast agent, by continuous infusion, said apparatus comprising (i) a delivery device adapted to retain a dispersion-containing delivery vessel in an essentially vertical position and controllably to expel dispersion from an upper or lower extremity of said vessel; (ii) mixing means adapted to effect admixture of said expelled dispersion with a flushing medium; and (iii) conduit means adapted to conduct said admixed dispersion and flushing medium to an administration device.

[0020] The term “essentially vertical” as used herein denotes that the longitudinal axis of the delivery vessel should be positioned within about 30E of vertical, preferably within 15E and more preferably within 5E of vertical. The vessel may be positioned for delivery of dispersion from either its upper or lower extremity, i.e. for upward or downward delivery respectively.

[0021] In the case of dispersions comprising a relatively low density dispersed phase, such flotation as may occur during administration of the dispersion will tend to lead to a reduction in dispersed phase concentration as administration proceeds in the case where the delivery vessel is positioned for upward delivery and to a corresponding increase in concentration in the case where the delivery vessel is positioned for downward delivery. It will be appreciated that the converse will apply for dispersions comprising a relatively high density dispersed phase which is susceptible to sedimentation. Such concentration changes may, if desired, be counteracted by appropriate adjustment of the rates at which the dispersion and flushing medium are coadministered. Additionally or alternatively the delivery vessel may be inverted at a suitable stage during infusion.

[0022] It is preferred that the delivery vessel is positioned so that the bulk flow direction of dispersion during expulsion is the same as the direction of segregation of the dispersed phase, since this will assist in counteracting the formation of concentration gradients of dispersed phase within the dispersion during administration. Thus, for example, in the case of dispersions such as gas-containing contrast agents in which the dispersed phase is susceptible to flotation, it is preferred to use delivery vessels positioned for upward delivery.

[0023] Delivery devices which may be used in apparatus according to the invention include syringe driver means such as power injection systems in which the syringe plunger is controllably driven by an appropriate automated mechanism, for example an electrically powered and controlled helical screw or push rod.

[0024] Where the infused dispersion is a gas-containing contrast agent it may, for example, be administered at a rate in the range 0.001-0.5 ml/minute, preferably 0.01-0.25 ml/minute, and may be selected to take account of factors such as the gas concentration and, in the case of ultrasound studies, the desired degree of attenuation. The infusion rate will depend on the body weight of the subject, and will typically be about 0.06:kg/hour. Such contrast agents may, for example, be administered over an infusion period of up to one hour, typically for a period of 15-20 minutes; steady state distribution of contrast agent in vivo will typically be achieved after 1-2 minutes

[0025] The flushing medium may be any appropriate biocompatible liquid, but is preferably normal (i.e. 0.9%) saline. It may, for example, be administered by gravitational flow using appropriate flow rate controlling means, or may be delivered using a controllable pump. Flow rates of 0.5-2 ml/minute, have been found to be appropriate although higher flow rates, e.g. up to 5 ml/minute, may also be useful.

[0026] Mixing of the dispersion and flushing medium may, for example, be effected in a three way connector, e.g. a T-piece, a Y-piece or a tap such as a three way stopcock, which is also connected via appropriate tubing to an administration device, e.g. an injection device such as a needle or catheter. It is preferred that connections are kept to a minimum and are made using low volume tubing in order to minimise transit time of the dispersion and thus to minimise the potential for segregation of the dispersed phase.

[0027] Gases which may be present in gas-containing contrast agents administered in accordance with the invention include any biocompatible substances, including mixtures, which are at least partially, e.g. substantially or completely, in gaseous or vapour form at the normal human body temperature of 37 EC. Representative gases thus include air; nitrogen; oxygen; carbon dioxide; hydrogen; inert gases such as helium, argon, xenon or krypton; sulphur fluorides such as sulphur hexafluoride, disulphur decafluoride or trifluoromethylsulphur pentafluoride; selenium hexafluoride; optionally halogenated silanes such as methylsilane or dimethylsilane; low molecular weight hydrocarbons (e.g. containing up to 7 carbon atoms), for example alkanes such as methane, ethane, a propane, a butane or a pentane, cycloalkanes such as cyclopropane, cyclobutane or cyclopentane, alkenes such as ethylene, propene, propadiene or a butene, and alkynes such as acetylene or propyne; ethers such as dimethyl ether; ketones; esters; halogenated low molecular weight hydrocarbons (e.g. containing up to 7 carbon atoms); and mixtures of any of the foregoing. Advantageously at least some of the halogen atoms in halogenated gases are fluorine atoms; thus biocompatible halogenated hydrocarbon gases may, for example, be selected from bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethylfluoride, 1,1-difluoroethane and perfluorocarbons. Representative perfluorocarbons include perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture with other isomers such as perfluoro-iso-butane), perfluoropentanes, perfluorohexanes or perfluoroheptanes; perfluoroalkenes such as perfluoropropene, perfluorobutenes (e.g. perfluorobut-2-ene), perfluorobutadiene, perfluoropentenes (e.g. perfluoropent-1-ene) or perfluoro-4-methylpent-2-ene; perfluoroalkynes such as perfluorobut-2-yne; and perfluorocycloalkanes such as perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethyl-cyclobutanes, perfluorocyclopentane, perfluoromethyl-cyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane or perfluorocycloheptane. Other halogenated gases include methyl chloride, fluorinated (e.g. perfluorinated) ketones such as perfluoroacetone and fluorinated (e.g. perfluorinated) ethers such as perfluorodiethyl ether. The use of perfluorinated gases, for example sulphur hexafluoride and perfluorocarbons such as perfluoropropane, perfluorobutanes, perfluoropentanes and perfluorohexanes, may be particularly advantageous in view of the recognised high stability in the blood stream of microbubbles containing such gases. Other gases with physicochemical characteristics which cause them to form highly stable microbubbles in the blood stream may likewise be useful.

[0028] Representative examples of contrast agent formulations include microbubbles of gas stabilised (e.g. at least partially encapsulated) by a coalescence-resistant surface membrane (for example gelatin, e.g. as described in WO-A-8002365), a filmogenic protein (for example an albumin such as human serum albumin, e.g. as described in U.S. Pat. Nos. 4,718,433, 4,774,958, 4,844,882, EP-A-0359246, WO-A-9112823, WO-A-9205806, WO-A-9217213, WO-A-9406477, WO-A-9501187 or WO-A-9638180), a polymer material (for example a synthetic biodegradable polymer as described in EP-A-0398935, an elastic interfacial synthetic polymer membrane as described in EP-A-0458745, a microparticulate biodegradable polyaldehyde as described in EP-A-0441468, a microparticulate N-dicarboxylic acid derivative of a polyamino acid - polycyclic imide as described in EP-A-0458079, or a biodegradable polymer as described in WO-A-9317718 or WO-A-9607434), a non-polymeric and non-polymerisable wall-forming material (for example as described in WO-A-9521631), or a surfactant (for example a polyoxyethylene-polyoxypropylene block copolymer surfactant such as a Pluronic, a polymer surfactant as described in WO-A-9506518, or a film-forming surfactant such as a phospholipid, e.g. as described in WO-A-9211873, WO-A-9217212, WO-A-9222247, WO-A-9409829, WO-A-9428780, WO-A-9503835 or WO-A-9729783). Contrast agent formulations comprising free microbubbles of selected gases, e.g. as described in WO-A-9305819, or comprising a liquid-in-liquid emulsion in which the boiling point of the dispersed phase is below the body temperature of the subject to be imaged, e.g. as described in WO-A-9416739, may also be used.

[0029] Other useful gas-containing contrast agent formulations include gas-containing solid systems, for example microparticles (especially aggregates of microparticles) having gas contained therewithin or otherwise associated therewith (for example being adsorbed on the surface thereof and/or contained within voids, cavities or pores therein, e.g. as described in EP-A-0122624, EP-A-0123235, EP-A-0365467, WO-A-9221382, WO-A-9300930, WO-A-9313802, WO-A-9313808 or WO-A-9313809). It will be appreciated that the echogenicity of such microparticulate contrast agents may derive directly from the contained/associated gas and/or from gas (e.g. microbubbles) liberated from the solid material (e.g. upon dissolution of the microparticulate structure). The invention may also be useful in conjunction with contrast agent systems based on microspheres comprising a therapeutic compound as described in e.g. WO-A-9851284 and WO-A-9927981.

[0030] The disclosures of all of the above-described documents relating to gas-containing contrast agent formulations are incorporated herein by reference.

[0031] Gas microbubbles and other gas-containing materials such as microparticles preferably have an initial average size not exceeding 10 :m (e.g. of 7 :m or less) in order to permit their free passage through the pulmonary system following administration, e.g. by intravenous injection. However, larger microbubbles may be employed where, for example, these contain a mixture of one or more relatively blood-soluble or otherwise diffusible gases such as air, oxygen, nitrogen or carbon dioxide with one or more substantially insoluble and non-diffusible gases such as perfluorocarbons. Outward diffusion of the soluble/diffusible gas content following administration will cause such microbubbles rapidly to shrink to a size which will be determined by the amount of insoluble/non-diffusible gas present and which may be selected to permit passage of the resulting microbubbles through the lung capillaries of the pulmonary system.

[0032] Where phospholipid-containing contrast agent formulations are employed in accordance with the invention, e.g. in the form of phospholipid-stabilised gas microbubbles, representative examples of useful phospholipids include lecithins (i.e. phosphatidylcholines), for example natural lecithins such as egg yolk lecithin or soya bean lecithin, semisynthetic (e.g. partially or fully hydrogenated) lecithins and synthetic lecithins such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine; phosphatidic acids; phosphatidylethanolamines; phosphatidylserines; phosphatidylglycerols; phosphatidylinositols; cardiolipins; sphingomyelins; fluorinated analogues of any of the foregoing; mixtures of any of the foregoing and mixtures with other lipids such as cholesterol. The use of phospholipids predominantly (e.g. at least 75%) comprising molecules individually bearing net overall charge, e.g. negative charge, for example as in naturally occurring (e.g. soya bean or egg yolk derived), semisynthetic (e.g. partially or fully hydrogenated) and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids and/or cardiolipins, for example as described in WO-A-9729783, may be particularly advantageous.

[0033] Representative examples of materials useful in gas-containing contrast agent microparticles include carbohydrates (for example hexoses such as glucose, fructose or galactose; disaccharides such as sucrose, lactose or maltose; pentoses such as arabinose, xylose or ribose; ″-, $- and (-cyclodextrins; polysaccharides such as starch, hydroxyethyl starch, amylose, amylopectin, glycogen, inulin, pulullan, dextran, carboxymethyl dextran, dextran phosphate, ketodextran, aminoethyldextran, alginates, chitin, chitosan, hyaluronic acid or heparin; and sugar alcohols, including alditols such as mannitol or sorbitol), inorganic salts (e.g. sodium chloride), organic salts (e.g. sodium citrate, sodium acetate or sodium tartrate), X-ray contrast agents (e.g. any of the commercially available carboxylic acid and non-ionic amide contrast agents typically containing at least one 2,4,6-triiodophenyl group having substituents such as carboxyl, carbamoyl, N-alkylcarbamoyl, N-hydroxyalkylcarbamoyl, acylamino, N-alkylacylamino or acylaminomethyl at the 3- and/or 5-positions, as in metrizoic acid, diatrizoic acid, iothalamic acid, ioxaglic acid, iohexol, iopentol, iopamidol, iodixanol, iopromide, metrizamide, iodipamicle, meglumine iodipamide, meglumine acetrizoate and meglumine diatrizoate), polypeptides and proteins (e.g. gelatin or albumin such as human serum albumin), and mixtures of any of the foregoing.

[0034] The method and apparatus of the invention may be particularly useful for infusion of the ultrasound contrast agents known as Levovist, Albunex, Optison, Definity, Imagent, Sonovue, Echogen, Sonogen and Sonazoid.

[0035] The method and apparatus of the invention may also be useful in sequential imaging procedures, for example in which a patient undergoes a first period of contrast agent infusion and imaging, is then subjected to stress (e.g. through exercise or by administration of a pharmacological stress agent such as adenosine, dobutamine, dipyridamole or arbutamine) and undergoes a second period of contrast agent infusion and imaging during or after this subjection to stress.

BRIEF DESCRIPTION OF DRAWINGS

[0036] In the accompanying drawings:

[0037]FIG. 1 is a schematic representation of one embodiment of apparatus useful in accordance with the invention;

[0038]FIG. 2 comprises plots of microbubble concentration against infusion time for the in vitro test system described in Example 6 hereinafter and for a comparative study using a horizontally positioned syringe; and

[0039]FIG. 3 comprises plots of echogenicity against time obtained in accordance with the in vivo studies described in Example 15 hereinafter.

[0040] Referring to FIG. 1 in more detail, syringe driver 1 (detail not shown) is adapted to receive vertically positioned syringe 2 and controllably to drive syringe plunger 3 in an upward direction so as to expel dispersion 4 through delivery outlet 5 at the upper extremity of the syringe. Three way stopcock 6 connects outlet 5 and feed 7 from saline infusion minibag 8 to conduit tube 9 which is connected via Luer lock 10 to infusion feed line 11, which in turn is connectable to an injection needle or catheter (not shown). The flow rate of dispersion is controllable by adjusting syringe driver 1. The flow rate of saline from minibag 8 is controllable by adjusting one or more of stopcock 6, valve 12 and the height of the minibag.

[0041] The following non-limitative examples serve to illustrate the invention.

Preparation 1—Hydrogenated Phosphatidylserine-encapsulated Perfluorobutane Microbubbles

[0042] Hydrogenated phosphatidylserine (5 mg/ml in a 1% w/w solution of propylene glycol in purified water) and perfluorobutane gas were homogenised in-line at 7800 rpm and ca. 40EC to yield a creamy-white microbubble dispersion. The dispersion was fractionated to substantially remove undersized microbubbles (C2 :m) and the volume of the dispersion was adjusted to the desired microbubble concentration. Sucrose was then added to a concentration of 92 mg/ml. 2 ml portions of the resulting dispersion were filled into 10 ml flat-bottomed vials specially designed for lyophilisation, and the contents were lyophilised to give a white porous cake. The lyophilisation chamber was then filled with perfluorobutane and the vials were sealed. Unless otherwise stated, 2 ml of water were added to a lyophilised product-containing vial prior to use and the contents were hand-shaken for several seconds, giving a perfluorobutane microbubble dispersion with a concentration range of 5-20×10⁸ microbubbles/ml (7-13 :l/ml).

EXAMPLE 1-6 In vitro Studies

[0043] The contents of vials prepared as in Preparation 1 were mixed with water (5 ml) from a syringe and gently hand shaken to give perfluorobutane microbubble dispersions with a microbubble concentration of about 3 :1/ml (Examples 1 to 5). In the procedure of Example 6 the contents of three vials were each mixed with 2 ml of water and the resulting dispersions were then pooled. Each of the thus-obtained microbubble dispersions was drawn into a syringe, which was vertically positioned in a module DPC syringe pump and connected to a low volume extension tube equipped with a 3 way stopcock and an administration set for delivery of normal saline from an infusion minibag, as shown in FIG. 1 The syringe pump rate and the saline rate were varied as shown in Table 1, which also records the calculated and observed microbubble concentrations and the observed periods over which steady state infusion was maintained (measured as timings from the start of infusion). TABLE 1 Example Syringe pump rate Saline flow rate Calculated microbubble Observed microbubble Period of steady state No. (ml/min) (ml/min) conc. (:1/ml) conc. (:1/ml) infusion (min) 1 0.017 1 0.05 0.11″ 0.04 10-60 2 0.1 1 0.3 0.29″ 0.05  5-30 3 0.2 1 0.6 0.57″ 0.06  5-16 4 0.017 2 0.025 0.08″ 0.01 10-60 5 0.1 2 0.15 0.17″ 0.03  5-30 6 0.1 2 0.35 0.44″ 0.04  5-35

[0044]FIG. 2 shows a plot of microbubble concentration (expressed as percentage of initial concentration) against time as determined in the procedure of Example 6. For comparison, the variation of microbubble concentration with time determined using an equivalent procedure with a horizontally positioned syringe is also shown. It can readily be seen that in the procedure according to the invention a substantially steady microbubble concentration is maintained, whereas in the comparative test the microbubble concentration rapidly decreases as infusion proceeds.

[0045] It will be appreciated that the length of the administration window will be shortened at higher syringe pump rates given the fixed volume of contrast agent present in a syringe.

EXAMPLES 7-14 In vitro Studies

[0046] In order to demonstrate the possibility of adjusting microbubble dose for different infusion procedures, a study was conducted according to the general procedure of Example 6 above, using a saline infusion rate of 2 ml/minute while varying the syringe pump rate as shown in Table 2, which also records the calculated and observed microbubble concentrations. TABLE 2 Calculated Observed Example Infusion Syringe pump microbubble microbubble No. time (min) rate (ml/min) conc. (:1/ml) conc. (:1/ml)  7  5 0.1  0.15  0.20  8 10 0.05 0.075 0.10  9 15 0.05 0.075 0.12 10 20 0.12 0.18  0.20 11 25 0.12 0.18  0.19 12 30 0.05 0.075 0.08 13 35 0.05 0.075 0.09 14 40 0.05 0.075 0.07

EXAMPLE 15 In vivo Study

[0047] Second harmonic imaging of the anterior myocardium was performed on an open chest model in 6 dogs (15-25kg, both sexes) using an ATL HDI 3000 scanner with a mechanical index of 0.6 and 1:4 triggering. Images were recorded following injection of a bolus of contrast agent prepared as in Preparation 1 at a concentration of 30 nl perfluorobutane/kg, and during infusion of contrast agent in accordance with the method of the invention at rates corresponding to 5, 15, 45 and 135 nl perfluorobutane/kg/min. The contrast effects in the region of interest are shown in FIG. 3.

EXAMPLE 16

[0048] A microbubble suspension is prepared as in Example 1 of WO-A-9748337 and administered according to the method of the invention.

EXAMPLE 17

[0049] The commercially available ultrasound product sold under the trade name Optison is administered according to the method of the invention.

EXAMPLE 18

[0050] Contrast agents are prepared as in Example 2 of WO-A-9927981 and administered according to the method of the invention.

EXAMPLE 19

[0051] Triggered ultraharmonic cardiac imaging of a patient with normal cardiac arteries was performed with triggering at every heart beat, every second heart beat, every fourth heart beat and every eighth heart beat. The heart was imaged from apical 2-chamber and 4-chamber views during dipyridamole-induced stress. Contrast agent prepared as in Preparation 1 was infused at a rate of 10 :1/kg/minute throughout the entire imaging period.

[0052] Increased videodensity was observed in each of the myocardial segments (the myocardium was divided into 16 segments according to the definitions of the American Society of Echocardiography) for each of the triggering intervals. The increase in intensity ranged from 8 to 10 dB in the different myocardial segments for the highest triggering interval (8 heart beats).

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195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu 340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 2 560 PRT Thermus aquaticus 2 Met Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu 1 5 10 15 Leu Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu 20 25 30 Gly Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala 35 40 45 Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala 50 55 60 Pro Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu 65 70 75 80 Leu Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu 85 90 95 Pro Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser 100 105 110 Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr 115 120 125 Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn 130 135 140 Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg 145 150 155 160 Glu Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr 165 170 175 Gly Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val 180 185 190 Ala Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly 195 200 205 His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe 210 215 220 Asp Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys 225 230 235 240 Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro 245 250 255 Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser 260 265 270 Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg 275 280 285 Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser 290 295 300 Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly 305 310 315 320 Gln Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val 325 330 335 Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser 340 345 350 Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His 355 360 365 Thr Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp 370 375 380 Pro Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Tyr Gly Val Leu Tyr 385 390 395 400 Gly Met Ser Ala His Arg Leu Ser Gln Arg Leu Ala Ile Pro Tyr Glu 405 410 415 Glu Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val 420 425 430 Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr 435 440 445 Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala 450 455 460 Arg Val Lys Ser Val Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 465 470 475 480 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 485 490 495 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 500 505 510 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 515 520 525 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 530 535 540 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 545 550 555 560 3 830 PRT Thermus aquaticus 3 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Ala Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu 195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu Ala Lys 340 345 350 Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro Pro Gly 355 360 365 Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr 370 375 380 Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu Glu Ala 385 390 395 400 Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly 405 410 415 Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu Val Glu 420 425 430 Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly Val Arg 435 440 445 Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala Glu Glu 450 455 460 Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His Pro Phe 465 470 475 480 Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp Glu Leu 485 490 495 Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr 500 505 510 Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val Glu 515 520 525 Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr Tyr Ile 530 535 540 Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu His Thr 545 550 555 560 Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp 565 570 575 Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln Arg Ile 580 585 590 Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala Leu Asp 595 600 605 Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly Asp Glu 610 615 620 Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr Glu Thr 625 630 635 640 Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met 645 650 655 Arg Arg Ala Ala Lys Thr Ile Asn Tyr Gly Val Leu Tyr Gly Met Ser 660 665 670 Ala His Arg Leu Ser Gln Arg Leu Ala Ile Pro Tyr Glu Glu Ala Gln 675 680 685 Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg Ala Trp 690 695 700 Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val Glu Thr 705 710 715 720 Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg Val Lys 725 730 735 Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro Val Gln 740 745 750 Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu Phe Pro 755 760 765 Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His Asp Glu 770 775 780 Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala Arg Leu 785 790 795 800 Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro Leu Glu 805 810 815 Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 

1. A method of administering a gravity segregating dispersion to a subject by continuous infusion, wherein said dispersion is controllably delivered from an upper or lower extremity of an essentially vertically positioned delivery vessel and thereafter is admixed with a flushing medium prior to administration to the subject.
 2. A method as claimed in claim 1 wherein said delivery vessel comprises a syringe.
 3. A method as claimed in claim 2 wherein delivery of said dispersion from said syringe is controlled by a syringe driver.
 4. A method as claimed in any of the preceding claims wherein said dispersion is a gas-containing contrast agent.
 5. A method as claimed in claim 4 wherein said gas comprises sulphur hexafluoride or a perfluorinated low molecular weight hydrocarbon.
 6. A method as claimed in claim 5 wherein said perfluorinated hydrocarbon is perfluoropropane or perfluorobutane.
 7. A method as claimed in any of claims 4 to 6 wherein said gas is present as albumin-stabilised microbubbles.
 8. A method as claimed in any of claims 4 to 6 wherein said gas is present as phospholipid-stabilised microbubbles.
 9. A method as claimed in claim 8 wherein said phospholipid predominantly comprises phosphatidylserine.
 10. A method as claimed in any of claims 4 to 9 wherein the delivery vessel comprises a syringe positioned for upward delivery of said contrast agent.
 11. A method as claimed in any of the preceding claims wherein said flushing medium is normal saline.
 12. A method as claimed in any of the preceding claims wherein the admixed dispersion and flushing medium are administered by injection.
 13. Apparatus for use in administration of a gravity segregating dispersion by continuous infusion, said apparatus comprising: (i) a delivery device adapted to receive a dispersion-containing delivery vessel in an essentially vertical position and controllably to expel dispersion from an upper or lower extremity of said vessel; (ii) mixing means adapted to effect admixture of said expelled dispersion with a flushing medium; and (iii) conduit means adapted to conduct said admixed dispersion and flushing medium to an administration device.
 14. Apparatus as claimed in claim 13 wherein said delivery device is a syringe driver adapted to receive an essentially vertically positioned syringe.
 15. Apparatus as claimed in claim 13 or claim 14 wherein said mixing means comprise a three way connector or tap adapted to connect said delivery vessel and a source of flushing medium to said conduit means.
 16. Apparatus as claimed in any of claims 13 to 15 which further comprises flow rate controlling means for controlling the rate of flow of said flushing medium.
 17. Apparatus as claimed in any of claims 13 to 16 which further comprises means for inverting the position of said delivery vessel.
 18. Use of apparatus as claimed in any of claims 13 to 17 in administration of a gravity segregating dispersion to a subject by continuous infusion. 